This invention relates to glycolipids and, more specifically, to glycosphingolipids and the synthesis of glycosphingolipids in substantially pure stereochemical form.
Glycosphingolipids are a class of complex molecules which are embedded in the membranes of cells throughout a wide variety of living organisms. Glycosphingolipids may be generically characterized as glycolipids because they are part lipid and part sugar; glycosphingolipids, however, constitute a special class of glycolipids in that they incorporate into their structures a class of lipids called ceramides, which comprise the lipid sphingosine and a fatty acid tail. Bound to the ceramide portion of the glycosphingolipid molecule is a carbohydrate chain. Variations in structure of both the ceramide and the carbohydrate are possible. In nature, however, certain carbohydrate structures are preferentially linked to certain ceramides. The number of naturally-occurring glycosphingolipids is apparently limited; only about 130 varieties of glycosphingolipids are now known.
That glycosphingolipids are found in all animal cells and in some plant cells has been known for some time. However, it is only recently that the biological function of these molecules has begun to be defined. Because glycosphingolipids are found on the membrane of a cell, it is believed that they help regulate the interactions of the cell with its environment. As a result of their regulatory function, glycosphingolipids are believed to play important roles in a number of serious diseases. It is thought that they may comprise infection receptor sites on the host cell for several kinds of viruses and bacteria.
Recent investigation has determined that brain and nervous tissues are rich in glycosphingolipids. The blood group antigens have also recently been identified as glycosphingolipids. The blood group antigens are found not only in blood but also in many kinds of tissue. For example, they are present in high concentrations on the surface of human epithelial cells, from which more than 90% of all human cancers are derived.
Because glycosphingolipids are present on these cancer prone epithelial cells, they have become objects of great interest by cancer immunologists and others. Immunological approaches to cancer treatment derive from the fact that the body responds to foreign antigens by the production of antibodies which selectively attack those antigens. Bacterial infections, for example, are commonly treated by vaccination with antibodies, similar to those produced by the body's own immune response, which attack antigens on the surface of the bacteria. It has been hypothesized that a cancerous tumor might similarly be treated with antibodies which would attack those antigens associated with the tumor. Many such tumor-associated antigens have been identified as glycosphingolipids; recent immunological studies have shown that many monoclonal antibodies directed against tumor-associated antigens are directed against glycosphingolipids. In addition, a number of studies have established that many types of antigens that are modified or inappropriately expressed in the development of malignant tumors are glycosphingolipids. For example, globotriaosylceramide (hereinafter, Gb.sub.3)--one of the most important members of this class of marker molecules--has been highly expressed in most Burkitt lymphoma cell lines, human teratocarcinoma, human embryonal carcinoma, and other types of tumor cells and has been shown to provide the cell surface receptor for verotoxin.
Thus research focusing on glycosphingolipids has been deemed to hold great promise in the treatment of disease, especially cancerous tumors. A considerable amount of work has been done to produce antibodies and reagents that specifically recognize and attack tumor-associated antigens such as glycosphingolipids. In principle, once the antibodies which attack particular tumor-associated antigens are identified, they can be produced in conventional ways and administered to cancer patients such as in a vaccine.
In identifying and growing such antibodies, it is desirable that the particular tumor-associated glycosphingolipid antigens to which the antibodies are directed be available as pure compositions. Commercial formulations of glycosphingolipids are presently available. However, such formulations are generally crude mixtures of a number of different glycosphingolipids and are obtained by involved and difficult extractions from animal and plant material. Synthetic approaches to pure glycosphingolipids in their naturally-occurring forms also have associated drawbacks. Because the glycosphingolipids are complex molecules, a given synthesized glycosphingolipid can comprise many isomeric forms; only one such isomer usually corresponds to the desired naturally-occurring glycosphingolipid, such as may comprise an antigen. Although stereoisomers, or enantiomers, are probably the most similar of all possible types of isomers, the relative reactivities of a naturally-occurring glycosphingolipid antigen and even its enantiomeric form would likely be widely disparate under conditions such as those found in mammals.
Synthetic routes to glycosphingolipids are known in the art, but such routes either provide the desired glycosphingolipid along with isomeric impurities which are removed only with difficulty or entail long experimental procedures which yield enantiomerically pure material only in low overall yield. For example, Schmidt, R. R. and Zimmermann, P., Angew. Chem. Int. Ed. Engl., 1986, 25, 725, teach the synthesis of a glycosphingolipid enantiomer in substantially pure form; the method employed therein involves the boron trifluoride catalyzed coupling of a D-glucosyl trichloroacetimidate with a benzoyl-protected azido derivative of D-erythro-C.sub.18 sphingosine. This method, however, produces the desired glycosphingolipid product in only about 56% overall yield from these intermediates.
It is therefore desired that short, efficient routes to substantially pure compositions of glycosphingolipids be available to facilitate the research and treatment of cancers and other diseases. Efficient means for synthesizing pure or substantially pure compositions of glycosphingolipids comprising carbohydrate/ceramide combinations not otherwise available from natural sources are also desired.
It is thus an object of this invention to provide synthetic routes to substantially pure glycosphingolipids. A further object is to provide such glycosphingolipids in substantially pure stereochemical form. Yet another object is to effect treatment of cancerous and other diseases by administering to a patient suspected of having the disease an antibody composition effective to result in reduction of the effects of the disease, said antibody composition being prepared using glycosphingolipids synthesized in accordance with this invention.